The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art in the present invention.
PCR is likely the most widely used method in modern molecular biology and biotechnology, and is rapidly being applied to genetic testing, diagnostics, forensics and biodefense. Kolmodin, L. A., et al., Nucleic Acid Protocols, 569-580 (2000); Budowle, B., et al., 301 Science, 1852-53 (2003); Y. Sato, et al., 5 (Suppl. 1) Legal Medicine, S191-S193 (2003); Saldanha, J., et al., 43 J. Medical Virol., 72-76 (1994); Dahiya, R., et al., 44 Biochemistry and Molecular Biology International, 407-15 (1998); and Elnifro, E. M., et al., 13 Clin. Microbiol. Rev., 559-70 (2000). PCR is described in U.S. Pat. Nos. 4,683,195 and 4,683,202. In each cycle of the PCR amplification process there are typically several steps. The double-stranded DNA target sequence is first thermally denatured at elevated temperatures (˜95° C.). The first occurrence of denaturation is referred to herein as the “initial denaturation step.” This is followed by annealing of a synthetic oligonucleotide primer to each strand at lower temperatures (˜60° C.). These forward and reverse oriented oligonucleotide primers are then each extended from their 3′ termini at an elevated temperature (˜70° C.) by a thermally stable, magnesium ion-dependent, DNA polymerase which incorporates 5′-deoxynucleotide triphosphates (dNTPs) and generates pyrophosphate (PPi), as depicted in the top portion of FIG. 1 for the forward oligonucleotide primer.
The utility of PCR is driven by its ability to rapidly provide target amplifications of ˜106-fold as well as high specificity, which depends in part on the specificity of oligonucleotide primer hybridization. Oligonucleotide primer sequences and length are therefore designed to hybridize to only the intended target sequence, at the temperatures used for annealing. However, PCR amplification reactions are typically prepared over a period of minutes or hours at ambient room temperatures which are well below the temperature range needed to ensure the specificity of oligonucleotide primer hybridization. Under such less stringent sample preparation conditions, the oligonucleotide primers may bind non-specifically to other sequences having substantial non-complementarity and potentially initiate the synthesis of undesired extension products, which can be amplified along with the target sequence. As has been discussed by Chou, Q., et al., amplification of non-specific sequences by this “mis-priming” can compete with amplification of the desired target sequences, and can therefore significantly decrease the efficiency of the amplification of the desired sequence, especially for low-copy number targets. Chou, Q., et al., 20 Nucleic Acids Res., 1717-23 (1992).
Formation of a “primer dimer” is another problematic form of non-specific hybridization, which, according to Chou, Q., et al., results from amplified extension of two oligonucleotide primers across one another's sequence without significant intervening sequence. These investigations further noted that primer dimers may undergo amplified oligomerization during PCR to create a complex mixture of oligonucleotide primer artifacts, the quality of which often varies inversely with the yield of specific PCR product in low copy number amplifications.
While the aforementioned problems due to mis-priming and primer dimer formation can be encountered in all applications of PCR, these issues can be particularly challenging for high-sensitivity analytical PCR schemes, such as those used for detection of blood-borne infectious agents (Saldanha, J., et al. and Elnifro, E. M., et al.), biohazardous microbes (Budowle, B., et al.) defective or cancerous genes (Dahiya, R., et al), and forensics (Budowle, B., et al. and Y. Sato, et al.). In addition, there is a much greater chance for formation of spurious amplification products in multiplex PCR. Markoulatos, P., et al., 16 J. of Clin. Laboratory Analysis, 47-51 (2002). In reverse transcriptase PCR (RT-PCR), the most sensitive means for detection of a target RNA sequence is to use a gene-specific oligonucleotide primer in the RT step. Zhang, J., et al., 337 Biochem. J., 231-41 (1999); Lekanne Deprez, R. H., et al., 307 Analytical Biochem., 63-69 (2002); and Bustin, S. A., et al., 15 J. of Biomolecular Techniques, 155-66 (2004). In view of the importance of these high-sensitivity applications requiring high specificity to avoid serious, adverse consequences of “false negatives” and “false positives”, it is critical to have reagents and protocols which provide assays that are functionally free of artifacts due to mis-priming and primer dimer formation.
A number of general strategies have been investigated for reducing non-specific PCR amplification based on the so-called “hot start” process which aims at impairing undesired amplification due to mis-priming and oligonucleotide primer dimer formation under low-stringency conditions at room temperature during sample preparation. PCR amplification subsequently begins when the amplification reaction mixture reaches high-stringency, “hot” temperatures to “start” polymerase-mediated extension of oligonucleotide primers hybridized only to target sequences. Thus temperature is used to trigger enzymatic extension of the oligonucleotide primers only at elevated temperatures when the stringency of primer/target hybridization conditions are optimal for specificity.
These general strategies for “hot start” include the use of (1) temperature-sensitive materials, such as waxes as barriers or sequestrants to control mixing of the reagents (Q. Chou, et al, and Tanzer, L. R., et al., 273 Anal. Biochem., 307-310 (1999)); (2) oligonucleotide aptamers (Dang, C., et al., 264 J. Mol. Biol., 268-78 (1996)) or antibodies (Eastlund, E., et al., 2 LifeScience Quarterly, 2-5 (2001) and Mizuguchi, H., et al., 126 J. Biochem (Tokyo), 762-68 (1999)) that inhibit the function of DNA polymerases; (3) use of a second thermostable enzyme, such as pyrophosphatase (Clark, D. R., et al., International Patent Application No. WO 2002088387) to remove suppression by added pyrophosphate (PPi); (4) chemically modified polymerases with hydrolytically reversible reagents, such as citraconic acid-modified lysine (Birch, D. E., et al., U.S. Pat. No. 5,773,258) in AmpliTaq Gold (Moretti, T., et al., 25 BioTechniques, 716-722 (1998) and Saldanha, J., et al.) and (5) oligonucleotide primer sequence constructs that disfavor low-temperature mis-priming, such as competitor sequences (Puskas, L. G., et al, 5 Genome Research, 309-311 (1995) or “touch-up and loop-incorporated oligonucleotide primers” (TULIPS-PCR) (Ailenberg, M., et al., 29(5) BioTechniques, 1018-23 (2000)).